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Modulation of Bleomycin-Induced Pulmonary Toxicity in the Hamster by l-Carnitine*
Linda Nici, MD
Barbara Monfils, BA
Paul Calabresi, MD
Department of Internal Medicine
The Rhode Island Hospital
and Brown University
Providence, Rhode Island
*The authors wish to thank Sigma-Tau for providing the l-carnitine used in this study.
KEY WORDS: bleomycin, lung fibrosis, antioxidant, l-carnitine, animal model
Background: Destruction of the alveolar epithelial barrier and subsequent intraalveolar fibroblast proliferation and deposition of extracellular matrix components characterize bleomycin pulmonary toxicity. Two crucial determinants in the development of pulmonary fibrosis are thought to be inability of type 2 pneumocytes to proliferate sufficiently to regenerate the injured or lost epithelial lining and unchecked fibroblast proliferation. Research has suggested an essential role for oxygen radicals in the pathogenesis of this process, perhaps in part, by influencing intracellular signaling events that control cell survival and death. l-carnitine has been shown to stabilize membranes and offer protection against free radicals. Our hypothesis is that l-carnitine can ameliorate bleomycin lung injury.
Methods: Hamsters (120-g) were given intratracheal (IT) bleomycin (1 unit) or saline, followed by daily IP l-carnitine or saline for 6 days. Lungs were assessed at day 21 for histologic assessment of fibrosis, biochemical analysis of lung collagen content, lung antioxidant levels, and lung cell apoptosis.
Results: No significant differences in morbidity or mortality were seen between groups. IT bleomycin, when compared to control animals, caused pulmonary fibrosis at day 21 as measured by morphometric analysis and increased hydroxyproline content. Animals treated with l-carnitine/bleomycin showed a significant decrease in percentage of fibrosis per lung as compared with bleomycin-injured animals (24.31% [+/-4.04] versus 41.28% [+/-6.32]) and hydroxyproline content (1.304 [+/-0.33] versus 2.104 [+/-0.217]). In addition, levels of the lung antioxidant, superoxide dismutase (SOD), measured via Western blotting, appear to be increased in animals cotreated with l-carnitine as compared to bleomycin alone. Finally, immunohistochemistry of whole lung tissue sections suggested increased proliferation and decreased apoptosis of alveolar epithelial cells in animals cotreated with l-carnitine/bleomycin as compared to bleomycin alone.
Conclusions: l-carnitine appears to provide a protective effect in the hamster model of bleomycin-induced lung injury as reflected in decreased areas of histologic fibrosis and reduced lung hydroxyproline content. This finding may be of clinical importance and warrants further study of possible mechanisms.
Bleomycin, an antimicrobial agent first described by Umezawa and coworkers,1 has been shown to be an effective antitumor agent in testicular carcinoma and lymphoma.2 In addition, it has also been used as cytotoxic therapy for other germ cell tumors, Kaposi's sarcoma, and head and neck carcinoma.3 A serious complication of bleomycin therapy is pulmonary fibrosis, which may occur in up to approximately 10% of patients to a variable degree.4 The cumulative dose of the drug, older age, radiation therapy, and supplemental oxygen therapy may increase the risk of pulmonary toxicity.5,6 In rodents, intratracheal instillation of bleomycin produces a reliable and useful model for the study of pulmonary fibrosis of various etiologies.7 Bleomycin-induced lung injury begins with an explosive inflammatory response in the alveolar wall. In the aftermath of tissue destruction, a fibroproliferative response ensues, leading to extensive intraalveolar granulation tissue comprised mainly of fibroblasts and their connective tissue products. Two crucial determinants in this pathologic process are thought to be inability of type 2 pneumocytes to proliferate sufficiently to regenerate the injured or lost epithelial lining and unchecked fibroblast proliferation.8 Active oxygen species (AOSs) generated in the lung by bleomycin are at least partially responsible for its toxic effects,6 as well as its therapeutic effects at the tumor site. AOSs are involved in a wide range of pathologic processes, including cytotoxicity, inflammation, and carcinogenesis.9
The carnitine system consists of l-carnitine, acetylcarnitines, and the cellular proteins required for their metabolism and transport.10 In mammals, the synthesis of carnitine is confined to the liver, the kidneys, the brain, and the testis.11 The carnitine system functions in the transport of activated short-, medium-, and long-chain fatty acids throughout the cells in the form of acetylcarnitine esters. The acetyl delivery is essential for mitochondrial ß-oxidation, membrane synthesis and repair, and the acetylation of proteins.12 Carnitine stabilizes membranes to offer protection against free radicals and has been shown to be protective in reperfusion after cardiac ischemia and to protect b-thalassemic erythrocytes from oxidative stress.13 Carnitine can also inhibit fas-induced apoptosis in lymphoma and leukemia cell lines.14 In addition, it has been shown to have neuroprotective effects, including inhibition of lipid-peroxidation and stimulation of antioxidant production.15 These effects have led to the concept of using carnitine supplementation in the aging process, dementia, protection against physical stress in top athletes, and protection against ischemic lesions. In light of these findings, we hypothesize that l-carnitine will protect lung tissue from the injury caused by bleomycin.
We now report that l-carnitine is able to significantly decrease fibrosis in the hamster model of bleomycin-induced lung injury. Our results suggest that the effects of l-carnitine may be partially due to an upregulation of endogenous antioxidants. Our results also suggest that l-carnitine may influence the ability of type 2 pneumocyte to repair the injured epithelial surface. These possible mechanisms will require further elucidation.
MATERIALS AND METHODS
Animal Model of Bleomycin-Induced Lung Injury: Adult male hamsters (approximately 120 g) were briefly restrained and anesthetized with intraperitoneal (IP) injection of sodium pentobarbitol (50 mg/kg). A 1-cm vertical incision was made in the skin over the trachea, and a 1-U solution of bleomycin (Bristol-Myers Squibb Co., Princeton, NJ) in 0.3 mL saline or 0.3 mL saline alone was instilled through a 27-gauge needle into the trachea while the animals were breathing spontaneously. The animals were rotated to distribute the solution, and the incisions closed. Animals were allowed to recover for 21 days under normal laboratory conditions with body weights measured every other day for the first week, then weekly until sacrificed with lethal IP injection of sodium pentobarbital. Lung tissue was prepared for histologic assessment, biochemical quantitation of hydroxyproline, Western blot analysis of the antioxidant superoxide dismutase, and histologic assessment of proliferation and apoptosis using BrdU incorporation and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay, respectively.
Cotreatment with l-carnitine: Animals were given IP injections of l-carnitine (gift of Sigma-Tau; Rome, Italy) at 200 mg/kg (1180 mg/m2) or equal volume saline, morning and evening (bid), on the day prior to intratracheal (IT) bleomycin and a third dose just prior to surgery. Animals were then given daily IP injections of l-carnitine or saline bid at the same dosage for a total of 7 days.
Experimental Groups: All studies were performed with 4 groups of animals, with 8 to 10 animals per group: (1) control animals, which received IP saline/IT saline; (2) animals that received IP saline/IT bleomycin; (3) animals that received IP l-carnitine/IT saline; (4) animals that received IP l-carnitine/IT bleomycin.
Histologic Assessment of Fibrosis
Morphology: Lungs were fixed by inflation at 20 cm H2O ex vivo with 10% formaldehyde in phosphobuffered saline (pH 7), embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Similarly oriented sections from the upper-, middle-, and lower-lobe portions of the lung were examined for the presence and degree of inflammatory infiltrate, interstitial and intraalveolar edema, and fibrosis.
Morphometry: Morphometry was performed on histologic sections prepared as described above using a computer-assisted color image processor equipped with a conventional light microscope. We determined the area of all fibrotic lesions in the sample sections as a percentage of the total profile of the lung sections (i.e., area of fibrosis-inflammation/total lung area).
Biochemical Quantitation of Fibrosis
Lung Hydroxyproline content: After lethal IP injection of pentobarbitol, hamster tracheobronchial tree and right ventricle were perfused with cold saline, then homogenized in phosphobuffered saline with protease inhibitors. Hydroxyproline content was performed by the method described by Reddy.17 Aliquots of lung homogenates were hydrolyzed in NaOH then autoclaved at 120°C for 20 minutes After addition of chloromine T, oxidation proceeded for 25 minutes, then Erlich's aldehyde reagent was added to allow chromophore to develop. Duplicate aliquots of each sample were assayed spectrophotometrically and compared with a standard assay curve using purified hydroxyproline. Hydroxyproline content is expressed as mg/lung.
Western Blot Analysis of Lung Superoxide Dismutase Levels
Lungs from anesthetized hamsters were perfused and lavaged with heparinized calcium and magnesium-free phosphobuffered saline, then minced and homogenized in 0.05 mol/L sodium phosphate buffer and centrifuged at 14,000 rpm for 10 minutes at 4°C. Supernatants were collected, aliquotted, and stored at -70°C until analysis. Twenty mg of total lung protein from each condition was applied to each lane, analyzed by 15% polyacrylamide sodium dodecyl sulfate (SDS) gel electrophoresis under reducing conditions, and transferred to nitrocellulose for Western blotting using primary anti-manganese superoxide dismutase (MnSOD; Calbiochem; San Diego, CA) and detection by enhanced chemiluminesence (ECL). The amount of MnSOD protein present in experimental animals was judged by comparison with the reaction product of the protein from uninjured animals present on the same membrane.
Determination of Cell Proliferation
Animals were injected intraperitoneally with 120 mg/kg of a 2.4% solution of BrdU in physiologic saline (Boehringer Mannheim, Indianapolis, IN) 2 hours prior to sacrifice. Hamsters were then anesthetized with a sublethal dose of pentobarbital. Their chests were opened, and their left ventricles were perfused with calcium and magnesium-free phosphobuffered saline at 20 cm H2O. Lungs were instilled with 4% paraformaldehyde for 10 minutes and then submerged in 4% paraformaldehyde until sectioned. Slices from the right middle lobe of the lung were paraffin embedded, sectioned (5 mm) onto glass slides, and stained with primary anti-BrdU IgG antibody (Oncogene Research Products, Cambridge, MA). Negative control slides were run with normal mouse IgG substituted for the primary antibody. Fc fragment-specific alkaline phosphatase-conjugated goat antimouse IgG (Jackson) was applied, followed by peroxidase-conjugated strepavidin (Biogenex) applied at a dilution of 1:10. Sections were counterstained with Mayer's hemotoxylin, mounted, and coverslipped. Cross reactivity and specificity was checked by running control sections in which antibody was omitted and all link and label antibodies were applied. Scoring of BrdU-labeled nuclei was determined by examining 100 random fields at 60x magnification and scoring the number of labeled nuclei.
Detection of Apoptosis
Paraffin sections of lung were dewaxed, rehydrated, and treated with proteinase K (20 mg/mL). Subsequent end-labeling with TdT (0.3 U/mL) in TdT buffer together with fluorescein dUTP was carried out for 1 hour at 37°C in a humidifying chamber. After end labeling, sections were washed in phosphobuffered saline, and analyzed by fluorescence microscopy. Apoptotic nuclei were identified by the presence of green fluorescent staining. Scoring of apoptotic nuclei was determined as described for BrdU-labeled nuclei.
All studies were performed a minimum of four times with all animal groups. Statistical analysis was performed by analysis of variance (Newman-Keuls post hoc test). Student's t-test of differences between means of paired samples was applied in Figure 1. Differences were considered statistically significant at P < .05.
Histologic Evaluation of Lung Injury/Fibrosis
Figure 1 illustrates the light microscopy of lung tissue obtained after inflation at 20 cm H2O ex vivo and photographed at the same magnification and exposure. (A) Lungs that received IP saline (not shown) or IP l-carnitine/IT saline showed normal alveolar architecture. (B) In animals that received IP saline/IT bleomycin, there was evidence of dense fibrosis, alveolar atelectasis, and edema. (C) In animals receiving IP l-carnitine/IT bleomycin, there were some areas of fibrosis, but they appear to be patchy with fewer areas of alveolar collapse.
Morphometric Evaluation of Lung Injury/Fibrosis
Table 1 illustrates the morphometric estimates of fibrosis for the 4 experimental groups. The quantitative estimates corresponded with our light microscopic findings. Control lungs and lungs given l-carnitine alone did not contain fibrotic areas. The amount of fibrosis in lungs treated with IP l-carnitine/IT bleomycin was significantly decreased when compared to the fibrosis seen with bleomycin alone.
Biochemical Quantitation of Lung Collagen
Table 2 illustrates the hydroxyproline content of lungs obtained from each experimental group. Hamsters that were untreated or given l-carnitine alone showed hydroxyproline levels consistent with established normal values. Animals injured with bleomycin showed an increase in hydroxyproline content consistent with increased collagen deposited during the fibrotic reaction. However, animals treated with IP l-carnitine/IT bleomycin had a significant decrease in hydroxyproline content compared with injured animals, consistent with amelioration of the fibrotic response.
Western Blot Analysis of Superoxide Dismutase Lung Antioxidant Levels:
Western blotting for MnSOD was performed on whole lung protein isolated from injured and control animals. Figure 2 shows a representative Western blot from 1 of 5 experiments. MnSOD is seen at 51 kDa in the positive control and in all experimental lanes. The amount of whole lung MnSOD appears to be increased after bleomycin-induced injury. There appears to be a further increase in whole lung MnSOD in animals cotreated with l-carnitine.
Determination of cell proliferation.
Detection of Apoptosis
Cleavage of genomic DNA during apoptosis yields double- and single-strand DNA breaks. Using a TUNEL assay, DNA strand breaks are identified by labeling free 3'-OH termini with modified nucleotides attached to a fluorescein label. In control (uninjured) animals, essentially no TUNEL staining is seen (Figure 4A). In animals that received IT bleomycin, there is evidence of apoptosis via TUNEL staining throughout the histologic sections examined (Figure 4B). In contrast, lung sections examined from animals that received IP l-carnitine/IT bleomycin show a reduction in the amount of TUNEL-positive cells, which may suggest a decrease in apoptosis (Figure 4C).
In this report, we have demonstrated that l-carnitine significantly decreases lung fibrosis after IT bleomycin. Lung fibrosis was assessed biochemically and by morphologic criteria. While histologic examination of lung tissue gives qualitative information, morphometry determines the area of fibrosis in histologic sections as a percentage of the total profile of the lung section. Although only a small fraction of lung tissue per animal was examined histologically, representative sections from upper-, middle-, and lower-lobe portions of the lung were used for morphometry in a minimum of 6 animals from 3 separate trials. To support our histologic findings, hydroxyproline content of the lung was determined with a standard biochemical assay. Hydroxyproline is a major constituent of collagen and has been used by a number of investigators to quantitate pulmonary fibrosis.16,18 Both morphometry and measurement of hydroxyproline show a significant decrease in lung fibrosis in bleomycin-injured animals treated with l-carnitine compared with injured animals alone.
Active oxygen species generated in the lung by bleomycin are at least partially responsible for its toxic effects.6 Therefore, the effects of l-carnitine on the acute inflammatory response are not surprising given its ability to protect various tissues from AOSs. Carnitine stabilizes membranes to offer protection against free radicals and has been shown to be protective in reperfusion after cardiac ischemia and to protect b-thalassemic erythrocytes from oxidative stress. In addition, l-carnitine has been shown to stimulate antioxidant production.10,12,13 In our bleomycin-induced injury model, l-carnitine may cause an upregulation of endogenous lung antioxidants similar to that reported in the CNS. Mammalian tissues contain 3 forms of SOD, an enzyme converting O2- to H2O2. One form of SOD containing manganese (MnSOD) is localized almost exclusively in the mitochondria and is selectively induced by cytokines, hyperoxia, and irradiation. Relative concentrations of MnSOD have been shown to increase preferentially in type 2 pneumocytes subsequent to inhalation of other oxidant-generating compounds such as asbestos and silica.19 However, in our model system, in addition to a possible increase in lung MnSOD levels, l-carnitine may also modulate the fibrotic response by affecting survival of cells crucial in the repair response.
Tissue homeostasis is maintained through a delicate balance between cell proliferation and cell death. A major biologic process tightly connected with such population control is the repair of injured tissue. It has been postulated that in survivors of acute lung injury, apoptosis functions as a prominent mechanism in the prompt and orderly elimination of intraalveolar mesenchymal cells.20 Bitterman and colleagues20 examined bronchoalveolar lavage fluid from patients meeting established criteria for adult respiratory distress syndrome both acutely (fewer than 3 days after injury) and chronically (more than 10 days after injury). They found that bronchoalveolar lavage fluid from patients during alveolar repair induced both fibroblast and endothelial cell death, while that obtained at the time of injury or from normal individuals did not. Conversely, inhibition of apoptosis in cell types crucial for normal function and repair would also be desirable. In HIV-infected individuals, the Fas/FasL pathway of apoptosis plays a role in CD4+ T cell depletion. The apoptotic signal through Fas involves the activation of sphingomyelinase, causing sphingomyelin breakdown and ceramide production. Carnitine administration to AIDS patients reduces Fas-induced apoptosis of these T cells and thereby may slow the progression of HIV disease.14 Similarly, l-carnitine may reduce apoptosis of type 2 pneumocytes after bleomycin injury.
In addition to effects on cell survival, l-carnitine may have antiinflammatory properties that can modulate the explosive inflammatory response to IT bleomycin. A reduction in the degree of acute lung injury caused by IT bleomycin should lead to a decrease in the resultant fibrosis. l-carnitine down-regulates tumor necrosis factor-a production, an important proinflammatory cytokine that can trigger cell death.13,15 l-carnitine has also been reported to improve lung surfactant production in fetal lungs due to its role in the pathway of membrane phospholipid turnover and remodeling. Destruction of surfactant, which promotes alveolar stability by reducing surface tension, is a consistent early event in acute lung injury by a multitude of stimuli.21
The studies presented here suggest at least 2 potential mechanisms for the effect of l-carnitine on bleomycin-induced lung injury. l-carnitine may upregulate endogenous antioxidant production and may inhibit apoptosis of type 2 pneumocytes, thereby allowing timely and efficient alveolar repair. However, these studies are qualitative in nature and will require further quantitative evaluation. Lung MnSOD mRNA levels as well as activity of the protein will provide further information on this antioxidant's role during injury and repair. Changes in survival of lung cells during bleomycin injury with and without l-carnitine administration will need to be evaluated quantitatively using flow cytometry and evaluation of other markers of the apoptotic process, such as PARP cleavage in isolated lung cells.
l-carnitine is a nutritional supplement with a wide safety profile; therefore, verifying a protective effect for this compound could translate quickly to the clinical setting. It will be necessary, however, to confirm the mechanisms of this purported protective effect in addition to ensuring no loss of the effectiveness of bleomycin as a chemotherapeutic agent.
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Figure 1. Light Microscopy. Representative light micrographs of lungs from experimental groups were photographed at the same magnification (x40) and exposure. (A) IP l-carnitine/IT saline; normal alveolar architecture. (B) IP saline/IT bleomycin; dense fibrosis, alveolar atelectasis, and edema. (C) IP l-carnitine/IT bleomycin; less dense areas of fibrosis with interspersed normal alveolar architecture.
Figure 2. Western Blot Analysis of Hamster Lung MnSOD. Lanes are identified at top, from right to left (MnSOD positive control; untreated animals; animals given IT bleomycin; animals given IT bleomycin and IP l-carnitine. Band visualized is at 39 kDa, the expected location of MnSOD. There is an apparent increase in MnSOD protein with IT bleomycin when compared with uninjured animals. There is a further increase in the amount of MnSOD protein in animals cotreated with bleomycin and l-carnitine.
Figure 3. BrdU Incorporation. Representative histologic lung sections from experimental animals injected with BrdU are shown. Cells with dark brown nuclei are positive for BrdU staining (arrows). (A) Untreated animals show normal lung architecture and rare BrdU positive nuclei. (B) Animals that received IT bleomycin show collapsed alveolar structures and occasionally BrdU-positive nuclei, which appear to be interstitial in location (arrows). (C) Animals that received IT bleomycin/IP l-carnitine show BrdU-positive nuclei on alveolar surfaces (arrows).
Figure 4. TUNEL. Immunofluorescence micrographs of TUNEL staining are shown for experimental groups. (A) Untreated animals rare TUNEL-positive staining. (B) Animals that received IT bleomycin have numerous areas of TUNEL-positive staining (arrows). (C) Animals that received IT bleomycin/IP l-carnitine have decreased areas of TUNEL-positive staining when compared with animals injured with IT bleomycin alone (arrows).
Table 1. Morphometric Comparison of Fibrosis in Bleomycin-Injured Lungs with and without l-carnitine Cotreatment
Fibrosis (mm2)/Profile area of lung (mm2) = area density of fibrosis (%)
Group (n = 6) Area Density (+/- SE)
1) Saline/saline 0%
2) Saline/bleomycin 41.28% (+/- 6.32)
3) l-carnitine/saline 0%
4) l-carnitine/bleomycin 24.31% (+/- 4.04)*
(P < .05)
Table 2. Comparison of Hydroxyproline Content (mg/lung) in Bleomycin-Injured Lungs with and without l-carnitine Cotreatment
Group (n = 6) Hydroxyproline (+/- SE)
1) Saline/saline 0.878 (+/- 0.063)
2) Saline/bleomycin 2.104 (+/- 0.217)
3) l-carnitine/saline 0.924 (+/- 0.025)
4) l-carnitine/bleomycin 1.304 (+/- 0.187)*
(P < .05)
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